Science China Life Sciences

Nucleotide-binding leucine-rich repeat (NLR) immune receptor genes are of significant value in disease resistance breeding and the control of viral diseases. Soybean mosaic virus (SMV) poses a serious threat to soybean production and the Rsv1 locus in soybean cultivar Suweon 97 confers broad-spectrum resistance against SMV strains G1 to G7; however, this locus harbors no fewer than 18 NLR genes, and thus the broad-spectrum antiviral mechanisms underlying the Rsv1 locus remain poorly understood to date. Here, we established a rapid and highly efficient screening system for cloning NLR genes from soybean Rsv1 locus and identified a broad-spectrum antiviral NLR gene 13g184900 from this highly complicated locus. The NLR encoded by 13g184900 can recognize viral P3 protein from all SMV strains (G1-G7) and another potyvirus Bean common mosaic virus (BCMV). The coiled-coil (CC) domain of this NLR directly interacts with viral P3 protein. Additionally, we showed that this NLR originated from wild soybean accession in East China and has been introduced into several soybean cultivars during domestication. Collectively, we developed a high-throughput screening system for identifying NLR genes in soybean and our study provides new mechanistic perspective on how the Rsv1 locus mediates the broad-spectrum resistance to all SMV G1-G7 strains.

Chikungunya virus (CHIKV) causes recurrent epidemics across tropical and subtropical regions globally. In 2025, Guangdong reported mainland China's largest documented CHIKV outbreak, with 23,464 cases across all 21 prefecture-level cities. Integrating epidemiological, genomic, and phylodynamic analyses, we investigated the outbreak's origins, transmission, and viral adaptation. The Guangdong strain belonged to the ECSA-MAL lineage, exhibiting a long internal branch that highlights significant global surveillance gaps. Phylodynamic modeling estimated viral introduction in early April 2025, revealing ~2.5 months of cryptic transmission alongside rising vector densities. Spatial case distribution was moderately associated with human mobility from the epicenters. Globally, phylogenetic analysis identified 33 potential adaptive mutations across nine proteins and 14 epidemic lineages, including validated and 15 novel mutations. Twelve novel mutations occurred in the Asian Urban lineage (AUL), predominantly affecting NSP3. This study underscores the need for enhanced pre-peak surveillance and continuous monitoring of viral adaptation across ecological regions.

Human norovirus (hNoV) is the leading global cause of acute gastroenteritis, imposing a substantial health and economic burden worldwide. Progress in understanding hNoV pathogenesis has been hindered by the lack of tractable small-animal models that recapitulate symptomatic infection. Although zebrafish larvae support hNoV replication, infection remains asymptomatic, limiting their utility for studying disease mechanisms and host-pathogen interactions. In this study, we report that the zebrafish embryo infection model, in which microinjection of hNoV at the early cell stage, resulted in robust systemic viral replication accompanied by overt pathological manifestations, including pericardial and renal edema, yolk and cranial opacity, and mortality by 3 days post-infection. Disease severity displayed marked individual variability and correlated closely with viral burden. Integrated multi-omics analyses, including bulk transcriptomics, untargeted metabolomics, and single-cell RNA sequencing, demonstrated that embryonic infection elicits a stronger and more coordinated antiviral response than larval-stage infection, while enabling widespread viral dissemination across diverse cell lineages. Approximately two-thirds of infected cells were derived from the nervous system or neural crest lineages, providing a potential mechanistic basis for the neurological complications occasionally reported in hNoV-infected patients. Furthermore, we identified a developmental stage-dependent role for extracellular vesicle (EV)-associated hNoV transmission: free virions mediated more efficient infection and higher symptomatic incidence in immunologically immature embryos, whereas EV-associated virions exhibited enhanced infectivity in more immunocompetent larvae. Together, these findings establish the zebrafish embryo as a versatile and accessible in vivo platform for studying symptomatic hNoV infection, reveal host maturity-dependent viral transmission strategies, and provide new opportunities for mechanistic studies and high-throughput evaluation of antiviral and vaccine candidates. Author summaryHuman norovirus is the leading cause of stomach flu worldwide but studying it has been difficult because the lack of a simple, small-animal model that actually gets sick from the virus. While older zebrafish larvae can harbor the virus, they do not show symptoms. In this study, we successfully created a new model by injecting human norovirus into zebrafish embryos at their early cell stage. Unlike the older larvae, these embryos developed clear symptoms, including fluid buildup around the heart and kidneys, tissue cloudiness, and death within three days. Using advanced genetic and metabolic tracking, we discovered that the virus spreads widely throughout the embryos body, particularly targeting cells in the nervous system. This link might explain why human patients occasionally suffer from neurological symptoms. Additionally, our study revealed that the virus changes its transmission strategy based on the animals age: it travels freely to infect vulnerable embryos but hides inside lipid vesicles to infect older, more immune-developed larvae. Ultimately, these findings provide a practical, efficient animal model to better understand norovirus sickness and rapidly test new vaccines and treatments.

Filovirus outbreaks caused by Ebola virus (EBOV) and Marburg virus (MARV), pose severe global health threats characterized by high rates of fatal hemorrhagic fever. While species-specific vaccines and therapeutic monoclonal antibodies are approved for Zaire ebolavirus, broadly-active therapeutics remain unavailable, leaving populations vulnerable to MARV and other pathogenic Ebola species, such as Bundibugyo (BDBV) and Sudan (SUDV) ebolaviruses. Here we report a computationally guided, infectious virus validated screening platform for the rapid discovery of broad-spectrum filovirus antivirals. By leveraging quantitative structure-activity relationship (QSAR) models, we screened 142,382 compounds in silico to prioritize 125 high-potential candidates. Subsequent dose-response and viability profiling identified 23 compounds exhibiting potent, low-micromolar pan-filovirus activity and favorable cytotoxicity profiles. Molecular docking indicates these compounds target conserved structural and functional domains--primarily the VP35 and L proteins--which may disrupt essential viral replication and immune antagonism. Furthermore, systematic combinatorial screening revealed three highly synergistic compound pairs, notably NCGC00113249-01 and NCGC00118008-01, demonstrating robust cross-species efficacy. By targeting conserved vulnerabilities across the filovirus family, this integrated in silico and in vitro pipeline provides a scalable framework to rapidly nominate and optimize synergistic therapeutic regimens against both endemic and emerging viral threats including BDBV. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/737586v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1251baorg.highwire.dtl.DTLVardef@b3a2feorg.highwire.dtl.DTLVardef@191d314org.highwire.dtl.DTLVardef@b8f710_HPS_FORMAT_FIGEXP M_FIG C_FIG

Chimeric antigen receptor (CAR)-T cell therapies have transformed the treatment of various tumor types by redirecting and activating T cells against tumor cells. However, CAR-T cell manufacturing approaches remain challenging and limit their widespread use in clinical settings. In vivo CAR-T therapy bypasses ex vivo cell manufacturing and patient preconditioning limitations; however, it faces a significant safety concern as CAR proteins on viral packaging cells are incorporated into budding virions, leading to off-target transduction of tumor cells. Here, we address this risk by developing the CAR-Less ER-Anchor Vector (CLEAN-V) system. By exploiting endoplasmic reticulum (ER) retention, CLEAN-V prevents the CAR protein from trafficking to the cell surface during viral packaging, thereby blocking its incorporation into the viral envelope. CLEAN-V particles exhibit near-complete loss of CAR-mediated tumor cell transduction. Furthermore, CLEAN-V integrates seamlessly into existing third-generation LVV workflows in four- or five-plasmid formats and generates CAR-T cells with preserved phenotypic and functional integrity. These results establish CLEAN-V as a robust platform for developing safe, targeted lentiviral vectors for in vivo CAR-T therapy.

Immunoglobulins (e.g., IgGs) are critical effectors of the adaptive immune system that when overexpressed or dysregulated can result in autoimmune diseases. Thus, depletion of IgGs can be a promising therapeutic avenue. Here we developed genetically-encoded lysosome targeting chimeras (GELYTACs) that target circulating IgGs for clearance and degradation. The GELYTACs comprised two protein modules derived from insulin-like growth factor 2 (IGF2) and an IgG-binding nanobody, respectively, and mediated clearance of plasma IgG via the lysosomal trafficking receptor IGF2R. To achieve long-lasting IgG depletion, we encoded GELYTACs in an AAV gene therapy vector and established continuous expression in mice. We also developed conditional GELYACs that are activatable with disease-specific proteases or small molecule drugs. This work establishes GELYTACs as a possible therapeutic modality that is deliverable using genetic medicine approaches.

Influenza virus poses a potential risk of triggering the next global pandemic. In-depth investigation into the mechanisms underlying influenza virus replication and pathogenicity will provide robust support for controlling influenza virus infection. Although post-translational modifications are known to regulate viral infection, the role of lactylation in influenza virus replication remains elusive. In this study, influenza virus ribonucleoprotein complex subunits are found to be lactylated. Specifically, ATAT1 promotes viral polymerase acidic protein (PA) lactylation and enhances viral replication. In contrast, SIRT1 mediates de-lactylation of PA and exerts an inhibitory effect on viral replication. Further investigations reveal lactylation of PA at residues K605 and K609 is essential for viral replication and pathogenicity. Mechanistically, PA K605/609 residues are localized at the interaction interface of the ANP32-mediated polymerase asymmetric dimer; mutation at these residues inhibits polymerase asymmetric dimerization, thereby impairing RNA production during viral genome replication. Collectively, this study uncovers a novel mechanism by which influenza virus hijacks host enzymes to mediate PA lactylation, and expands the molecular regulatory network of influenza virus infection.

The lack of effective anti-hepatitis B virus (HBV) therapies highlights the need for a new type of treatment that targets different stages of the viral life cycle. The HBV core protein (HBc) is a critical component of this cycle. Various capsid assembly modulators (CAMs) have been developed to target the HBc and inhibit HBV replication. We recently described a subset of capsid assembly modulators (CAMs) that induce the formation of aberrant structures from the HBc in the nucleus, leading to cell death via annexin A1 (ANXA1)-driven apoptosis. Thus, we further elucidated the mechanism of HBc aggregation in the nucleus, with a particular focus on the interplay between nuclear HBc aggregates and PML nuclear bodies. We found that long-term treatment with CAM-A induced the formation of enlarged PML bodies, approximately 1-2 m in diameter, that accumulated aggregated HBc. PML silencing in HBc-overexpressing HepG2-NTCP cells led to a dramatic increase in apoptosis following CAM-A-induced HBc aggregation, which was associated with elevated ANXA1. Next, we showed that PML nuclear bodies orchestrate proteasomal degradation of nuclear HBc aggregates via sumoylation-dependent recruitment of RNF4. Collectively, our results suggest that PML nuclear bodies act as storage compartments for aggregated HBc proteins in the nucleus, thereby counteracting the apoptotic elimination of cells. Further study of PML function and the targeting of PML nuclear bodies in HBV-infected hepatocytes could reveal new ways to enhance the effectiveness of CAMs.

Camptothecin derivatives are first-line anticancer drugs used worldwide for the treatment of diverse malignant tumors. However, the biosynthetic pathway of camptothecin has remained elusive for five decades. Here, we fully map its entire biosynthetic route. We discovered five key missing enzymes (OpCAR, OpSDR11, OpCS, OpGH1, and OpSTR) via the combination of MALDI mass spectrometry imaging, single-cell RNA sequencing and co-expression analysis. Meanwhile, we demonstrated a free flavin mononucleotide triggered the non-enzymatic 6-5-6 to 6-6-5 fused-ring skeleton rearrangement, filling the last gap in camptothecin biosynthesis. Finally, we validated this identified pathway and achieved the de novo biosynthesis of camptothecin in Saccharomyces cerevisiae. These discoveries uncover the long-standing mystery underlying camptothecin and pave the way for manufacturing camptothecin and its derivatives through synthetic biology approaches.

IgG-degrading enzymes have emerged as innovative therapeutic agents for treating conditions driven by pathogenic antibodies. Here, we used structure-guided rational design to engineer IdeSM33, a double mutant (K167R/D226E) of the IgG-specific bacterial protease IdeS from Streptococcus pyogenes, with improved catalytic efficiency. Biolayer interferometry revealed a fourfold increase in binding affinity relative to wild-type IdeS (IdeSWT). This enhancement is likely attributable to mutations that strengthen hydrogen bonding at the enzyme-IgG Fc interface. In vitro, IdeSM33 has higher performance than IdeSWT in cleaving serum IgG. In vivo studies in rabbits demonstrated that IdeSM33 effectively depleted circulating IgG and showed better performance at a dose of 0.005 mg/kg than the IdeSWT. Although doses greater than 0.2 mg/kg demonstrated higher plasma concentrations of IdeS and a larger AUC 0 to last, they did not show a significant enhancement in the pharmacodynamics of IgG degradation. Importantly, a single dose of IdeSM33 (0.2 mg/kg) potently degraded binding and neutralizing antibodies against AAV9 within 1-2 days and restored hepatic AAV9 transduction in pre-immunized animals. Together, these findings highlight IdeSM33 as a potent and safe engineered enzyme with therapeutic potential for autoimmune disorders, transplant rejection, and overcoming pre-existing humoral immunity in gene therapy.

Bunyamwera virus (BUNV) is the prototypical member of the Peribunyaviridae family of arthropod-borne viruses and possesses a genome comprising three segments of negative-sense RNA, named small, medium and large. The medium segment encodes a polyprotein that is processed to form Gn and Gc spikes and a non-structural protein, NSm. The role of NSm during replication in mammalian cells is poorly characterized, although it associates with a Golgi-derived structure called the virus factory (VF), the site of BUNV genome replication and virion assembly. To further define NSm function, we generated an epitope-tagged BUNV and used co-immunoprecipitation and quantitative proteomics to identify host interacting partners. NSm interacted with BCL-2 interacting protein 1 (BNIP1), a SNARE protein involved in COPI vesicle trafficking, with the importance of this interaction demonstrated by siRNA-mediated knockdown of BNIP1 expression, which significantly reduced BUNV gene expression and virion production. Interestingly, NSm also interacted with components of the NRZ complex, involved in COPI vesicle tethering in association with BNIP1, and inhibition of COPI complex formation resulted in loss of NSm expression. Taken together, our results identify BNIP1 as a host cell factor necessary for efficient BUNV replication and suggest the cellular localization of NSm at the VF is COPI-dependent.

The intestine plays a crucial role in physiology, nutrition, and immune function, but intestinal anatomy and cell types have yet to be fully characterized in many fish species, the most diverse group of vertebrates. To address this gap, we characterized the structure and composition of the intestine of threespine stickleback (Gasterosteus aculeatus), an emerging model teleost in biological research. Using histology, myeloperoxidase staining, single-cell RNA sequencing, and RNA in situ hybridization, we defined major intestinal epithelial, immune, stromal, and stem/progenitor populations. Goblet cells were abundant in proximal and hindgut, while myeloperoxidase-positive granulocytes were evenly distributed throughout the intestine. To facilitate future experimental studies of stickleback intestinal function, we also developed the first intestinal organoid culture from stickleback and show that these cultures recapitulate epithelial architecture and retain expression of canonical intestinal epithelial markers. This organoid platform enables future functional studies of mucosal immunity, host-microbe interactions, and intestinal physiology in stickleback and related teleosts. Together, our integrated approach provides a comprehensive cell atlas and a novel experimental model for studying digestive and immune functions in threespine stickleback.

Brain organoids offer an invaluable model system for studying human brain development and disease. However, the establishment of high-fidelity brain organoids with multiple cell lineages including vasculature and immune cells remains a huge challenge. Here, we present a new strategy to generate human cerebral organoids with vasculature and microglia-like cells using genetic code expansion technology (GCE-T) via site-specific protein engineering. The strategy integrates orthogonal genetic translation machinery in hPSCs via PiggyBac transposon system, enabling temporally control of ETV2 expression and endothelial differentiation in hPSC-derived cerebral organoids. The vascularized human cerebral organoids (vhCOs) exhibit coordinated development of multiple cell lineages and blood-brain barrier (BBB) features. Moreover, vhCOs form perfusable vascular network after transplanted in the immune-deficient mice. Single-nucleus RNA sequencing reveals enhanced neurovascular interactions, multi-brain-regional identities, diverse neuronal subtypes and specialized endothelial subclusters in vhCOs, closely resembling human fetal brain. Strikingly, we identify enriched microglia-like cells comprising three distinct subtypes in vhCOs, which contribute to microglia-vascular interactions and synergistically modulate vascular development. Upon Zika virus (ZIKV) infection, vhCOs show neurovascular dysfunction and impaired microglia development, offering new insights into viral-induced neurodevelopmental disorders. This study offers a unique platform for producing more valuable brain organoids with vasculature and immune components, opening a new avenue to advance organoid research and applications.

Here, we present the first graph-based Korean Pangenome Reference (K-PanRef), constructed from 14 healthy Korean individuals. K-PanRef comprises 13 high-quality diploid Korean genome assemblies (mean QV ~62.0) and KOREF1-G-TTAGGA, the first complete Korean reference genome. Integration of these assemblies generated a ~3.2-Gb pangenome graph containing ~39.3 million nodes and ~53.8 million edges, with the accumulation of common sequences (frequency [≥]10%) reaching a plateau. Additionally, K-PanRef contains ~4.3 million Korean-specific small variants and ~76.0 thousand Korean-specific SVs absent from the Chinese and human pangenome references, improving the representation of Korean genetic diversity relative to these references. To evaluate its utility for short-read-based SV analysis, we genotyped 75 whole-genome sequencing (WGS) samples, including 15 patients with early-onset myocardial infarction (MI). Although constructed entirely from healthy genomes, K-PanRef supported the identification of putative disease-relevant SVs in this exploratory application. K-PanRef-based genotyping identified ~95.6 thousand small variants and 820 SVs observed only in the early-onset MI samples. Among the early-onset MI-group SVs, 491 were absent from public databases, suggesting that they may represent previously unrecognized candidate variants related to early-onset MI. Of these, 164 SVs overlapped 134 genes, of which 89 had reported associations with 42 cardiovascular diseases or traits, including eight genes previously linked to MI. Together, these results establish K-PanRef as a valuable resource for representing Korean genetic diversity and enabling more comprehensive discovery of population-specific and novel putative disease-relevant variants from short-read sequencing data.

Cas12a2 CRISPR nucleases, including SuCas12a2, have been shown to have extensive collateral activity towards RNA, ssDNA, and dsDNA. This collateral activity results in targeted cell elimination and has applications across biotechnology, agriculture, and human health. We explored the natural genetic diversity of Cas12a2 nucleases and characterized nine novel orthologs in a DNA damage kinetic assay in E. coli. Three new Cas12a2 orthologs (RsCas12a2, SdCas12a2, and HmCas12a2) were shown to have high collateral activity towards DNA. These nucleases are highly divergent from SuCas12a2, have conserved core RuvC catalytic residues, and have sequence diversity in the previously reported aromatic clamp residues required for nucleic acid positioning in the active site. We defined PFS preferences and mismatch tolerance for each high-activity Cas12a2 nuclease, expanding the available Cas12a2 toolbox, and discovered functional differences with obvious impacts on downstream applications.

Zika virus (ZIKV) is an arthropod-borne flavivirus of international public health impact. ZIKV has a positive-sense, single-stranded RNA genome and remodels intracellular membranes to form replication complexes (RCs). The objective of this study was to isolate and characterize the RCs from ZIKV-infected cells and to identify host-cell components recruited to participate in viral replication. Here, we isolated the RCs from ZIKV-infected Vero cells by detergent treatment and flotation centrifugation. Fractional flotation analysis demonstrated that ZIKV proteins NS2B, NS3, and NS5, and ZIKV RNA were present in the detergent-resistant membranous fraction. In contrast, the ER-resident protein calnexin and a mitochondrial protein were present in the detergent-soluble fractions. The isolated RCs were functional for ZIKV RNA synthesis, as shown by quantitative PCR. To determine the components of the RCs, we conducted mass spectrometry analysis and identified numerous cellular proteins. Among them is the replication factor C subunit 2 (RFC2), an accessory protein of DNA polymerase. RFC2 is involved in ATP binding and hydrolysis and may promote cell survival. ZIKV infection increased the RFC2 protein level and induced its relocation to the cytoplasm. RNAi-mediated silencing of RFC2 reduced ZIKV replication. Together, our results provide insights into ZIKV replication and virus-cell interaction.

Predicting linear B-cell epitopes is a basic immunoinformatics task that has a direct impact on vaccine design and antibody engineering. Recent advances in machine learning have improved predictive performance, but most existing approaches are trained on aggregated datasets and assume that antigenic patterns are conserved across host organisms. This assumption ignores the immunological variability depending on the host and prevents generalizing the model across species. This is the first systematic host-wise evaluation where we present a systematic machine learning-based analysis of host-aware linear B-cell epitope prediction using curated datasets from the Immune Epitope Database (IEDB). We build separate datasets for human, mouse, and non-human primate hosts and assess several classification models, including Random Forest, Support Vector Machine (SVM), Gradient Boosting, XGBoost, and K-Nearest Neighbors (KNN). The models exploit feature representations derived from sequences, such as AAIndex descriptors, biochemical properties from ExPASy, and dipeptide composition. Our results show that predictive performance differs substantially across hosts. Models achieve up to 86.07% accuracy and 0.93 ROC-AUC on human datasets but lower performance on mouse and non-human primate datasets. This gap underlies dataset bias and sequence distribution differences, as well as the inability of existing features to capture host-specific immunological context. These results indicate that the prediction of linear B-cell epitopes is intrinsically host-specific, and a single global model does not generalize well across species. We propose to incorporate host-aware modeling strategies and organism-specific features for enhanced predictive reliability and biological relevance.

Retroviruses are excellent tools for delivering and expressing transgenic cargoes with broad utility in research and therapy. However, many cargoes including virus restriction factors can dramatically limit virus production and/or infectivity. An extreme viro-toxic cargo is the DNA cytosine deaminase APOBEC3B, which potently restricts retrovirus infectivity by a direct cDNA deamination-dependent mechanism. To overcome viro-toxicity, an APOBEC3B minigene cargo is disrupted by a translation stop cassette flanked by a direct repeat of its own sequence. The integrity of the minigene is reconstituted naturally by retroviral recombination during transduction. Efficiency can be improved from 90% to nearly 100% by coupling the minigene to the translation of a downstream selectable marker. Collapsing retrovirus (CRV) technology enables the functional delivery of APOBEC3B to a target cell population and may have broad utility for delivering viro-toxic cargoes.

Capacitation, the process whereby sperm gain the functional competence to fertilize an egg in the absence of de novo transcription and translation, is orchestrated by a hierarchy of kinases driving the phosphorylation of sperm proteins. While increased phosphorylation, in particular tyrosine phosphorylation, is a revered hallmark of fertilization competency in our species, only a limited repository of phosphorylated substrates and kinases have ever been reported from human sperm. To broaden therapeutic targets for sperm targeted contraceptives and infertility therapies, we adapted a contemporary phosphoproteomic technique termed EasyPhos to generate bespoke methodology for the investigation of human sperm signalling. This approach yielded high depth phosphoproteomes of non-capacitated and capacitated human spermatozoa with in silico investigation of the phosphosites revealing 52 kinases with previously uncharacterized roles in sperm capacitation. Investigating the function of the putative sperm capacitation kinases identified yielded several kinases with novel roles in the regulation of sperm function. Of particular interest, polo like kinase 1 (PLK1) inhibition significantly reduced progressive sperm motility, attenuated capacitation-associated tyrosine phosphorylation and reduced the sperm acrosome reaction, an essential step to achieve fertilization. These findings reveal extensive phosphoproteome remodelling during human sperm capacitation, expanding the landscape of molecular targets for fertility control.

Abstract The Duroc-derived reference genome Sscrofa11.1 has provided a critical foundation for pig genomics, providing a high-quality reference genome for accurate variant detection and comparative genomics but does not capture breed-specific variation. Here, we present a near-complete, gap-free genome assembly for the Landrace pig (Landrace_v1, GCA_963921485.1), spanning all 20 chromosomes and totaling 2.6 Gb, including 176 Mb of sequence absent from Sscrofa11.1. Comparative analyses with recently published high-quality pig genomes reveal a conserved centromere organization across breeds, accompanied by substantial variation in repeat composition and length, and identify a pig specific pattern of telomere variant repeats across eight pig breeds. The improved resolution of repetitive regions in Landrace_v1 enables more complete reconstruction of complex gene families, including olfactory receptors, and uncovers structural variation at the KIT proto-oncogene receptor tyrosine kinase locus not represented in the Duroc reference. Together, these findings highlight the limitations of single-reference genomes and demonstrate the value of breed-specific assemblies for capturing genomic diversity and improving downstream analyses.